Comparison of cell death and adenosine triphosphate content as indicators of acute toxicity in vitro

1. Anchorage-independent LS cells, derived from L929 mouse fibroblasts, were used as an in vitro alternative to animals for the assessment of acute toxicity. The two end points were cell death, indicated by fluorescein diacetate and ethidium bromide, and intracellular adenosine triphosphate (ATP) content. 2. Concentrations of 20 test compounds which produced a 50% decrease in the ATP contents of control cells (ATP50) ranged from 17 micrograms ml-1 for diethylstilboestrol to 7.0 mg ml-1 for sodium chloride. 3. The concentrations which caused 50% cell death ranged from 16 micrograms ml-1 for diethylstilboestrol to 8.0 mg ml-1 for paracetamol. 4. There was a good numerical correlation (r = 0.99) between the ranks of ATP50 and CD50 end-points, there being only minor changes in order between the ranks. 5. The slopes of the dose-response plots for individual chemicals were markedly different.     

Deoxyribonucleoside triphosphate pools and adenosine toxicity

The toxic effect of adenosine on the metabolism of malignant lymphoid cells has been studied in relation to the activity of intracellular adenosine deaminase. Exposure in vitro of L1210 and L5178Y cells for 48 hr to adenosine demonstrated that concentrations above 10^?5 M inhibited cell division, the toxic effect being inversely proportional to intracellular adenosine deaminase levels. Measurement of the deoxyribonucleoside triphosphate pools in cells exposed to adenosine resulted in a 22 per cent reduction in the pyrimidine deoxyribonucleoside triphosphates. Adenosine-mediated growth inhibition was markedly enhanced by coformycin, a potent inhibitor of adenosine deaminase.     

外源性三磷酸腺苷和腺苷对肿瘤细胞增殖的影响

目的　研究三磷酸腺苷 (ATP)和腺苷 (ADO)对人红白血病细胞株K5 6 2、人胃中分化腺癌细胞株HGC 2 7、人食管低分化鳞癌细胞株TE 13细胞增殖的影响。方法　采用MTT法测定ATP和ADO抑制肿瘤细胞增殖的作用 ,Wright’s Giemsa染色观察细胞形态学的改变。结果　在 0 0 1～ 1 0mmol·L-1浓度范围内 ,ATP和ADO可不同程度地抑制TE 13、HGC 2 7和K5 6 2细胞的增殖 ,其中对TE 13的作用最强 ;ATP和ADO作用 72h后 ,对TE 13细胞的抑制率分别为80 5 2 %和 74 0 3%。细胞经高浓度 (1mmol·L-1)的ATP和ADO处理后 ,细胞形态出现了凋亡的特征。结论　ATP抗TE 13和HGC 2 7肿瘤细胞增殖的作用与其代谢产物ADO部分相关 ,ATP和ADO的作用机制可能涉及细胞凋亡     

Cardiotonic steroids: correlation of sodium-potassium adenosine triphosphate inhibition and ion transport in vitro with inotropic activity and toxicity in dogs.

1. A new series of cardiotonics based on five steroid nuclei has been evaluated for inhibition of Na-+/K-+-ATPase and Rb uptake by red blood cells, and for inotropic activity and toxicity in dogs. Structure-activity relationships are discussed. 2. The in vitro tests can be used satisfactorily to predict inotropic activity, but not toxicity or therapeutic ratio. 3. Although compounds with greatly improved therapeutic ratios relative to ouabain and tolusin have been obtained, they proved to be strongly emetic in the conscious dog.     

In vitro approaches to evaluate palytoxin-induced toxicity and cell death in intestinal cells

Palytoxin isolated from the genus Palythoa is the most potent marine toxin known. The aim of the present study was to quantify palytoxin-induced cellular injury in the human intestinal cell line Caco-2. Cellular damage was measured by evaluating cell proliferation, cell membrane permeability, cell morphology and apoptotic markers. Furthermore, changes in F-actin were studied after exposure of cells to increasing amounts of palytoxin. The results show that cell proliferation decreased in a concentration-dependent manner with a mean IC(50) value of about 0.1 nM. A noticeable increase of cell detachment correlated with cell rounding and F-actin depolymerization was observed in palytoxin-treated cells. Moreover LDH was released from the cells in a dose and time dependent manner, although under these conditions there was no propidium iodide uptake. On the other hand, palytoxin impaired mitochondrial activity but other apoptotic markers, such as DNA fragmentation or caspases activation, were not observed. The results obtained in this paper suggest that the effects of palytoxin in Caco-2 cells were very potent and unspecific, since a primary necrosis and a secondary apoptosis seem to occur under these conditions.     

Soil respiration,ATP content,and Photobacterium toxicity test as indicators of metal pollution in soil

The effects of heavy metal deposition from four different metalworks on soil respiration, ATP content, and toxicity to Photobacterium phosphoreum were examined. Soil samples were collected along a transect through the pollution gradients. Vegetation and soil types were homogeneous throughout the transects. Soil respiration was measured as CO₂ production in closed bottles using an IR-gas analyzer. The ATP content was measured with a bioluminometric assay after trichloracetic acid extraction of soil. The toxicities of distilled water extracts of soil samples were evaluated by the standard P. phosphoreum toxicity test and the metal content of these water extracts was measured as the bioavailable fraction of the heavy metal pollution in the soil. Soil respiration and ATP content were strongly affected by the heavy metal content in the soil. The correlation between the methods was high. The P. phosphoreum procedure can be used to estimate the toxicity of soils heavily polluted by metals. However, soil respiration and ATP content appear to be more sensitive indicators of soil pollution. © 1994 by John Wiley & Sons, Inc..     

Effects of adenosine, adenosine triphosphate and structural analogues on glucagon secretion from the perfused pancreas of rat in vitro

The effects of adenosine, adenosine triphosphate (ATP) and structural analogues have been studied on glucagon secretion from the isolated perfused pancreas of the rat in the presence of glucose (2.8 mM). Adenosine induced a transient increase of glucagon secretion. This effect was concentration-dependent in the range of 0.165 to 165 microM. ATP also induced an increase, but the effect was no greater at 165 microM than at 16.5 microM. 2-Chloroadenosine, an analogue more resistant to metabolism or uptake systems than adenosine, was more effective. Among the three structural analogues of ATP or ADP studied, beta, gamma-methylene ATP which can be hydrolyzed into AMP and adenosine had an effect similar to adenosine or ATP at the same concentrations (1.65 and 16.5 microM); in contrast alpha, beta-methylene ATP and alpha, beta-methylene ADP (resistant to hydrolysis into AMP and adenosine) were ineffective. Theophylline (50 microM) a specific blocker of the adenosine receptor, suppressed the glucagon peak induced by adenosine, 2-chloroadenosine, ATP and beta, gamma-methylene ATP (1.65 microM). An inhibitor of 5' nucleotidase, alpha, beta-methylene ADP (16.5 microM), reduced the glucagon increase induced by ATP and did not affect the response to adenosine (1.65 microM). These results support the hypothesis of adenosine receptors (P1-purinoceptors) on the pancreatic glucagon secretory cells and indicate that ATP acts after hydrolysis to adenosine.     

三磷酸腺苷或腺苷对心律失常的诊疗作用

三磷酸腺苷或腺苷是临床诊疗心律失常的一种重要方法，它可终止阵发性室上性心动过速，终止特殊类型室速，对多种类型的心律失常有鉴别诊断作用。另外，三磷酸腺苷或腺苷对窦房结功能障碍也有诊断价值，还可应用于射频导管消融术。     

Cyclic adenosine monophosphate-dependent vascular responses to purinergic agonists adenosine triphosphate and uridine triphosphate in the anesthetized mouse.

The mechanism by which purinergic agonist adenosine triphosphate (ATP) and uridine triphosphate (UTP) decrease systemic arterial pressure in the anesthetized mouse was investigated. Intravenous injections of adenosine triphosphate (ATP) and uridine triphosphate (UTP) produced dose-dependent decreases in systemic blood pressure in the mouse. The order of potency was ATP > UTP. Vasodilator responses to ATP and UTP were altered by the cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor rolipram. The vascular responses to ATP and UTP were not altered by a nitric oxide synthase inhibitor, a cyclooxygenase inhibitor, a cGMP phosphodiesterase inhibitor, or a particular P2 receptor antagonist. These data suggest that ATP and UTP cause a decrease in systemic arterial pressure in the mouse via a cAMP-dependent pathway via a novel P2 receptor linked to adenylate cyclase and that nitric oxide release, prostaglandin synthesis, cGMP, and P2X1, P2Y1, and P2Y4 receptors play little or no role in the vascular effects of these purinergic agonists in the mouse.     

The effects of adenosine triphosphate and related purines on arterial resistance vessels in vitro and in vivo

The distribution of P2-purinoceptors in pre-capillary resistance vessels was studied in vitro, using Krebs perfused rabbit ears and in vivo, in autoperfused hindquarters, intestinal and renal vasculatures of pentobarbitone anaesthetised cats. ATP (10(-10)-10(-6) mol i.a.) caused dose-dependent vasodilatation which, in the rabbit ear, was antagonised by reactive blue 2 (10(-5)-10(-4) M). At the highest concentration of reactive blue 2, ATP responses were reversed and a dose-dependent vasoconstriction was seen. Reactive blue 2, also reduced the vasodilator responses to carbachol and to a lesser extent papaverine which suggests that the antagonist has limited selectivity. The rank order of potency of ATP analogues as vasodilators, 2-methylthio ATP greater than ADP greater than ATP greater than alpha,beta-methylene and beta,gamma-methylene ATP, suggests P2y purinoceptors are involved. The selective P2x-purinoceptor agonist, alpha,beta-methylene ATP, caused pronounced vasoconstriction in the rabbit ear and cat intestinal vasculature which was not antagonised by phenoxybenzamine. In contrast, alpha,beta-methylene ATP had little effect in the autoperfused hindquarters and renal vasculatures suggesting a very heterogeneous distribution of P2x-purinoceptors in the cat. The results are consistent with the proposal that two distinct types of P2-purinoceptors are present on blood vessels.     

The effect of non-steroidal anti-inflammatory drugs on adenosine triphosphate content and histamine release from rat peritoneal cell suspensions rich in mast cells.

1 Non-steroidal anti-inflammatory drugs (NSAID) suppressed compound 48/80-induced histamine release from rat peritoneal cells in vitro in a dose-dependent manner. 2 NSAID suppressed the adenosine triphosphate (ATP) content of rat peritoneal cells in vitro and this correlated strongly with the suppression of compound 48/80-induced histamine release. 3 The correlation demonstrated suggests that the mechanism of action of NSAID in the rat peritoneal cells is via depletion of cellular ATP.     

不同品种桉叶制备的桉叶止咳糖浆体外抑菌活性和急性毒性比较

目的 比较两种不同品种桉叶制备的桉叶止咳糖浆体外抑菌活性和急性毒性,为临床用药提供理论依据.方法 采用纸片琼脂扩散法(K-B法),比较两种桉叶止咳糖浆体外抑菌活性.采用小鼠灌胃给药测定小鼠对不同品种的桉叶止咳糖浆的最大给药量,比较两种桉叶止咳糖浆的急性毒性.结果 用大叶桉和蓝桉分别制备的桉叶止咳糖浆对卡它莫拉菌(包括产β-内酰胺酶菌株)、流感嗜血杆菌(包括产β-内酰胺酶菌株)、肺炎链球菌产生的抑菌圈直径依次为(24.37±2.54)mm、(12.07±1.46)mm、(19.30±0.96)mm和(19.77±2.33)mm、(9.19±1.12)mm、(14.10±1.13)mm.测得小鼠对不同品种桉叶止咳糖浆最大给药量为192g(生药)/kg,相当于人临床1d用量的480倍.结论 大叶桉制备的桉叶止咳糖浆体外抑菌活性优于蓝桉制备的桉叶止咳糖浆,且两者毒性均较低,口服安全无毒,临床用药较为安全.     

In vitro potential measurement, anaesthetic and antimicrobial effects as indicators of beta-blocker toxicity of the cornea

Three tests were utilized to determine and compare the toxicity of timolol, propranolol and two new aliphatic and alicyclic oxime ethers with beta-blocking activity (falintolol and compound POS 7). Effects on the electrical potential difference across the in vitro bovine corneal epithelium. Local anaesthesia on in vivo rabbit cornea. Antimicrobial activity on bacterial and fungal suspensions. In addition, partition coefficients were determined as physicochemical properties of the drugs. Falintolol, as well as timolol produced a minor change in electrophysiology at clinical concentration. They had neither local anaesthetic, nor antimicrobial effects. Conversely, propranolol and compound POS 7 showed acute corneal toxicity in the present models. It was concluded that changes in the potential difference across a perfused cornea in vitro, local anaesthesia and bacterial inhibition, might be a demonstration of the cytotoxicity of certain topical agents in terms of acute eye tissue reaction. They might represent a valuable model for the acute corneal toxicity evaluation of topical beta-blockers.     

Testicular toxicity in vitro: Sertoli-germ cell co-cultures as a model system

Testicular damage is a comparatively common finding in toxicity studies. Part of the susceptibility of the testis to chemical toxicity stems from the complexity of its normal structure, function and regulatory control. This complexity has stimulated the development and extensive use of testicular cell cultures in the study of normal testicular function. However, the potential of such systems for investigating chemically induced testicular toxicity has only recently been considered. Using primary co-cultures of Sertoli and germ cells many features of the well characterized testicular damage produced by phthalate esters and glycol ethers could be reproduced in vitro. These encouraging findings are discussed in relation to some of the likely limitations to the application of in vitro models for testicular toxicity.     

注射用三磷酸腺苷二钠及其注射液的稳定性研究

目的研究注射用三磷酸腺苷二钠及其注射液的稳定性,并将两者的稳定性进行比较。方法将三磷酸腺苷二钠冻干品和注射液各3批进行加速试验和长期试验,期间测定各项指标,分别与起始数据比较,同时将冻干品和注射液的含量变化进行对比。结果加速试验及长期试验期间,各项指标都符合标准,但冻干品与注射液在试验过程中含量随时间的变化存在显著性差异。结论注射用三磷酸腺苷二钠稳定性优于三磷酸腺苷二钠注射液,有效期可定为2年。     

Effects of cadmium acetate and sodium selenite on mucociliary functions and adenosine triphosphate content in mouse trachea organ cultures

The effects of cadmium acetate and sodium selenite in mouse trachea organ culture have been studied separately and in combination. Ciliary activity, morphology, rate of total protein and glycoconjugate (i.e. glycoprotein and proteoglycan) synthesis/secretion and ATP content were investigated. Exposure to 10 microM cadmium acetate or 2000 microM sodium selenite resulted in a complete cessation of ciliary activity within 5 h. With cadmium acetate also a swelling of epithelial cells was observed. Sodium selenite (250-2000 microM) delayed by 2-3 h the inhibitory effect of cadmium acetate (5-20 microM) on ciliary activity. The rate of protein synthesis, as determined by incorporation of [3H]proline, was reduced by 13% and 44% at exposure for 4 h at 37 degrees C to 250 microM and 500 microM sodium selenite respectively, the effect being partly abolished by cadmium acetate. With 5 microM and 10 microM cadmium acetate the rate of glycoconjugate synthesis, as measured by incorporation of [3H]glucosamine, increased by 50% and 69%, respectively, after incubation for 4 h. This increase was partly reduced by sodium selenite. Neither cadmium acetate nor sodium selenite had any effect on the rate of total protein or glycoconjugate secretion. The ATP content in trachea rings was reduced by 48% and 54% after incubation for 4 h with 250 microM and 500 microM sodium selenite, respectively. No significant effect of cadmium acetate was found on ATP content. An antagonistic effect of sodium selenite and cadmium acetate in mouse trachea organ culture is suggested from the present experiments.